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1.  Copy Number Variation in CNP267 Region May Be Associated with Hip Bone Size 
PLoS ONE  2011;6(7):e22035.
Osteoporotic hip fracture (HF) is a serious global public health problem associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of key measurable risk factors for HF, independent of bone mineral density (BMD). Hip BS is highly genetically determined, but genetic factors underlying BS variation are still poorly defined. Here, we performed an initial genome-wide copy number variation (CNV) association analysis for hip BS in 1,627 Chinese Han subjects using Affymetrix GeneChip Human Mapping SNP 6.0 Array and a follow-up replicate study in 2,286 unrelated US Caucasians sample. We found that a copy number polymorphism (CNP267) located at chromosome 2q12.2 was significantly associated with hip BS in both initial Chinese and replicate Caucasian samples with p values of 4.73E-03 and 5.66E-03, respectively. An important candidate gene, four and a half LIM domains 2 (FHL2), was detected at the downstream of CNP267, which plays important roles in bone metabolism by binding to several bone formation regulator, such as insulin-like growth factor-binding protein 5 (IGFBP-5) and androgen receptor (AR). Our findings suggest that CNP267 region may be associated with hip BS which might influence the FHL2 gene downstream.
doi:10.1371/journal.pone.0022035
PMCID: PMC3137628  PMID: 21789208
2.  Pathway-Based Genome-Wide Association Analysis Identified the Importance of Regulation-of-Autophagy Pathway for Ultradistal Radius BMD 
Journal of Bone and Mineral Research  2010;25(7):1572-1580.
Wrist fracture is not only one of the most common osteoporotic fractures but also a predictor of future fractures at other sites. Wrist bone mineral density (BMD) is an important determinant of wrist fracture risk, with high heritability. Specific genes underlying wrist BMD variation are largely unknown. Most published genome-wide association studies (GWASs) have focused only on a few top-ranking single-nucleotide polymorphisms (SNPs)/genes and considered each of the identified SNPs/genes independently. To identify biologic pathways important to wrist BMD variation, we used a novel pathway-based analysis approach in our GWAS of wrist ultradistal radius (UD) BMD, examining approximately 500,000 SNPs genome-wide from 984 unrelated whites. A total of 963 biologic pathways/gene sets were analyzed. We identified the regulation-of-autophagy (ROA) pathway that achieved the most significant result (p = .005, qfdr = 0.043, pfwer = 0.016) for association with UD BMD. The ROA pathway also showed significant association with arm BMD in the Framingham Heart Study sample containing 2187 subjects, which further confirmed our findings in the discovery cohort. Earlier studies indicated that during endochondral ossification, autophagy occurs prior to apoptosis of hypertrophic chondrocytes, and it also has been shown that some genes in the ROA pathway (e.g., INFG) may play important roles in osteoblastogenesis or osteoclastogenesis. Our study supports the potential role of the ROA pathway in human wrist BMD variation and osteoporosis. Further functional evaluation of this pathway to determine the mechanism by which it regulates wrist BMD should be pursued to provide new insights into the pathogenesis of wrist osteoporosis. © 2010 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.36
PMCID: PMC3153999  PMID: 20200951
osteoporosis; bone mineral density; genome-wide association; regulation of autophagy; whites
3.  Genome-Wide Association Study for Femoral Neck Bone Geometry 
Poor femoral neck bone geometry at the femur is an important risk factor for hip fracture. We conducted a genome-wide association study (GWAS) of femoral neck bone geometry, examining approximately 379,000 eligible single-nucleotide polymorphisms (SNPs) in 1000 Caucasians. A common genetic variant, rs7430431 in the receptor transporting protein 3 (RTP3) gene, was identified in strong association with the buckling ratio (BR, P = 1.6 × 10−7), an index of bone structural instability, and with femoral cortical thickness (CT, P = 1.9 × 10−6). The RTP3 gene is located in 3p21.31, a region that we found to be linked with CT (LOD = 2.19, P = 6.0 × 10−4) in 3998 individuals from 434 pedigrees. The replication analyses in 1488 independent Caucasians and 2118 Chinese confirmed the association of rs7430431 to BR and CT (combined P = 7.0 × 10−3 for BR and P = 1.4 × 10−2 for CT). In addition, 350 hip fracture patients and 350 healthy control individuals were genotyped to assess the association of the RTP3 gene with the risk of hip fracture. Significant association between a nearby common SNP, rs10514713 of the RTP3 gene, and hip fracture (P = 1.0 × 10−3) was found. Our observations suggest that RTP3 may be a novel candidate gene for femoral neck bone geometry. © 2010 American Society for Bone and Mineral Research
doi:10.1359/jbmr.090726
PMCID: PMC3153387  PMID: 20175129
genome-wide association; femoral neck bone geometry; bone fracture; RTP3
4.  Genome-wide association scans identified CTNNBL1 as a novel gene for obesity 
Human Molecular Genetics  2008;17(12):1803-1813.
Obesity is a major public health problem with strong genetic determination; however, the genetic factors underlying obesity are largely unknown. In this study, we performed a genome-wide association scan for obesity by examining approximately 500 000 single-nucleotide polymorphisms (SNPs) in a sample of 1000 unrelated US Caucasians. We identified a novel gene, CTNNBL1, which has multiple SNPs associated with body mass index (BMI) and fat mass. The most significant SNP, rs6013029, achieved experiment-wise P-values of 2.69 × 10−7 for BMI and of 4.99 × 10−8 for fat mass, respectively. The SNP rs6013029 minor allele T confers an average increase in BMI and fat mass of 2.67 kg/m2 and 5.96 kg, respectively, compared with the alternative allele G. We further genotyped the five most significant CTNNBL1 SNPs in a French case–control sample comprising 896 class III obese adults (BMI ≥ 40 kg/m2) and 2916 lean adults (BMI < 25 kg/m2). All five SNPs showed consistent associations with obesity (8.83 × 10−3 < P < 6.96 × 10−4). Those subjects who were homozygous for the rs6013029 T allele had 1.42-fold increased odds of obesity compared with those without the T allele. The protein structure of CTNNBL1 is homologous to β-catenin, a family of proteins containing armadillo repeats, suggesting similar biological functions. β-Catenin is involved in the Wnt/β-catenin-signaling pathway which appears to contribute to maintaining the undifferentiated state of pre-adipocytes by inhibiting adipogenic gene expression. Our study hence suggests a novel mechanism for the development of obesity, where CTNNBL1 may play an important role. Our study also provided supportive evidence for previously identified associations between obesity and INSIG2 and PFKP, but not FTO.
doi:10.1093/hmg/ddn072
PMCID: PMC2900891  PMID: 18325910
5.  Plasmacytoid dendritic cells induce NK cell–dependent, tumor antigen–specific T cell cross-priming and tumor regression in mice 
The Journal of Clinical Investigation  2008;118(3):1165-1175.
A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-γ production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8+ T cells.
doi:10.1172/JCI33583
PMCID: PMC2230660  PMID: 18259609
6.  Copy Number Variations at the Prader–Willi Syndrome Region on Chromosome 15 and associations with Obesity in Whites 
Obesity (Silver Spring, Md.)  2011;19(6):1229-1234.
Obesity is a serious health problem with strong genetic determination. Copy number variation (CNV) is a common type of genomic variant associated with some complex human diseases. However, it is not clear how CNVs contribute to the etiology of obesity. In this study, we examined 1,000 unrelated US whites to search for CNVs that may predispose to obesity. We focused our analyses on the Prader–Willi syndrome (PWS) critical region (chromosome 15q11–q13), because the PWS region is a hotspot for CNV generation and obesity is one of the major clinical manifestations for chromosome abnormalities at this region. We constructed a map containing 39 CNVs at the PWS critical region with CNV occurrence rates higher than 1%. Among them, three CNVs were significantly associated with body fat mass (P < 0.05), with a higher copy number (CN) associated with an increase of 5.08–9.77 kg in body fat mass. These three CNVs are close to two known PWS genes, NDN (necdin homolog) and C15orf2 (chromosome 15 open reading frame 2), and partially overlap with another obesity gene PWRN1 (Prader–Willi region nonprotein-coding RNA 1). Interestingly, our recently published whole genome association scan study using the same sample by examining single-nucleotide polymorphisms (SNPs) did not find any significant associations at these CNV regions, suggesting the importance of examining both CNVs and SNPs for better understanding of genetic basis of obesity. Further studies are warranted to validate these CNVs and their importance to obesity.
doi:10.1038/oby.2010.323
PMCID: PMC4512297  PMID: 21233802
7.  Is GSN Significant for Hip BMD in Female Caucasians? 
Bone  2014;63:69-75.
Low bone mineral density (BMD) is a risk factor of osteoporosis. Osteoporosis is more prevalent in the females than the males. So far, the pathophysiological mechanisms underlying osteoporosis are unclear. Peripheral blood monocytes (PBM) are precursors of bone-resorbing osteoclasts. This study aims to identify PBM-expressed proteins (genes) influencing hip BMD in humans.
We utilized three independent study cohorts (N=34, 29, 40), including premenopausal Caucasians with discordant hip BMD. We studied PBM proteome-wide protein expression profiles in Cohort 1 and identified 57 differentially expressed proteins (DEPs) between low vs. high BMD subjects. One protein gelsolin (GSN), after validation by Western blotting, was subject to follow-up. We compared GSN mRNA level in PBM between low vs. high BMD subjects in Cohorts 2 and 3. We genotyped SNPs across GSN in 2,286 unrelated Caucasians (Cohort 4) and 1,627 Chinese (Cohort 5), and tested association with hip BMD in the females and males, respectively.
We discovered and validated that GSN protein expression level in PBM was down-regulated 3.0-fold in low vs. high BMD subjects (P<0.05). Down-regulation of GSN in PBM in low BMD subjects was also observed at mRNA level in both Cohorts 2 and 3. We identified that SNP rs767770 was significantly associated with hip BMD in female Caucasians (P=0.0003) only. Integrating analyses of the datasets at DNA, RNA, and protein levels from female Caucasians substantiated that GSN is highly significant for hip BMD (P=0.0001).
We conclude that GSN is a significant gene influencing hip BMD in female Caucasians.
doi:10.1016/j.bone.2014.02.015
PMCID: PMC4127973  PMID: 24607942
Bone mineral density; Monocyte; Gelsolin; Integration analysis
8.  Genome-wide association study identifies HMGN3 locus for spine bone size variation in Chinese 
Human genetics  2011;131(3):463-469.
Bone size (BS) is one of the major risk factors for osteoporotic fractures. BS variation is genetically determined to a substantial degree with heritability over 50%, but specific genes underlying variation of BS are still largely unknown. To identify specific genes for BS in Chinese, initial genome-wide association scan (GWAS) study and follow-up replication study were performed. In initial GWAS study, a group of 12 contiguous single-nucleotide polymorphism (SNP)s, which span a region of ~ 25 kb and locate at the upstream of HMGN3 gene (high-mobility group nucleosomal binding domain 3), achieved moderate association signals for spine BS, with P values ranging from 6.2E–05 to 1.8E–06. In the follow-up replication study, eight of the 12 SNPs were detected suggestive replicate associations with BS in 1,728 unrelated female Caucasians, which have well-known differences from Chinese in ethnic genetic background. The SNPs in the region of HMGN3 gene formed a tightly combined haplotype block in both Chinese and Caucasians. The results suggest that the genomic region containing HMGN3 gene may be associated with spine BS in Chinese.
doi:10.1007/s00439-011-1093-7
PMCID: PMC4450081  PMID: 21947420
9.  The anti-fibrotic effects of mesenchymal stem cells on irradiated lungs via stimulating endogenous secretion of HGF and PGE2 
Scientific Reports  2015;5:8713.
Radiation-induced pulmonary fibrosis is a common disease and has a poor prognosis owing to the progressive breakdown of gas exchange regions in the lung. Recently, a novel strategy of administering mesenchymal stem cells for pulmonary fibrosis has achieved high therapeutic efficacy. In the present study, we attempted to use human adipose tissue-derived mesenchymal stem cells to prevent disease in Sprague-Dawley rats that received semi-thoracic irradiation (15 Gy). To investigate the specific roles of mesenchymal stem cells in ameliorating radiation-induced pulmonary fibrosis, we treated control groups of irradiated rats with human skin fibroblasts or phosphate-buffered saline. After mesenchymal stem cells were infused, host secretions of hepatocyte growth factor (HGF) and prostaglandin E2 (PGE2) were elevated compared with those of the controls. In contrast, tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta1 (TGF-β1) levels were decreased after infusion of mesenchymal stem cells. Consequently, the architecture of the irradiated lungs was preserved without marked activation of fibroblasts or collagen deposition within the injured sites. Moreover, mesenchymal stem cells were able to prevent the irradiated type II alveolar epithelial cells from undergoing epithelial-mesenchymal transition. Collectively, these data confirmed that mesenchymal stem cells have the potential to limit pulmonary fibrosis after exposure to ionising irradiation.
doi:10.1038/srep08713
PMCID: PMC4348621  PMID: 25736907
10.  The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells 
In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.
doi:10.1038/cmi.2013.40
PMCID: PMC4002151  PMID: 24037184
DHX33; helicase; innate immunity; IPS-1; myeloid dendritic cell; viral nucleic acid
11.  SNP rs6265 Regulates Protein Phosphorylation and Osteoblast Differentiation and Influences BMD in Humans 
Bone Mineral Density (BMD) is major index for diagnosing osteoporosis. PhosSNPs are non-synonymous SNPs that affect protein phosphorylation. The relevance and significance of phosSNPs to BMD and osteoporosis is unknown. This study aims to identify and characterize phosSNPs significant for BMD in humans. We conducted a pilot genome-wide phosSNP association study for BMD in three independent population samples, involving ~5,000 unrelated individuals. We identified and replicated three phosSNPs associated with both spine BMD and hip BMD in Caucasians. Association with hip BMD for one of these phosSNPs, i.e., rs6265 (major/minor allele: G/A) in BDNF gene, was also suggested in Chinese. Consistently in both ethnicities, individuals carrying AA genotype have significant lower hip BMD than carriers of GA and GG genotypes. Through in vitro molecular and cellular studies, we found that compared to osteoblastic cells transfected with wild-type BDNF-Val66 (encoded with allele G at rs6265), transfection of variant BDNF-Met66 (encoded with allele A at rs6265) significantly decreased BDNF protein phosphorylation (at amino acid residue T62), expression of osteoblastic genes (OPN, BMP2, and ALP), and osteoblastic activity. The findings are consistent with and explain our prior observations in general human populations. We conclude that phosSNP rs6265, via regulating BDNF protein phosphorylation and osteoblast differentiation, influence hip BMD in humans. This study represents our first endeavor to dissect the functions of phosSNPs in bone, which might stimulate extended large-scale studies on bone or similar studies on other human complex traits and diseases.
doi:10.1002/jbmr.1997
PMCID: PMC4127979  PMID: 23712400
BMD; SNP; protein phosphorylation; BDNF; osteoblast
12.  Genome-Wide Association Study Identified UQCC Locus for Spine Bone Size in Humans 
Bone  2012;53(1):129-133.
Bone size (BS) contributes significantly to the risk of osteoporotic fracture. Osteoporotic spine fracture is one of the most disabling outcomes of osteoporosis. This study aims to identify genomic loci underlying spine BS variation in humans.
We performed a genome-wide association scan in 2,286 unrelated Caucasians using Affymetrix 6.0 SNP arrays. Areal BS (cm2) at lumbar spine was measured using dual energy X-ray absorptiometry scanners. SNPs of interest were subjected to replication analyses and meta-analyses with additional two independent Caucasian populations (N = 1,000 and 2,503) and one Chinese population (N = 1,627).
In the initial GWAS, 91 SNPs were associated with spine BS (P<1.0E-4). Eight contiguous SNPs were found clustering in a haplotype block within UQCC gene (ubiquinol-cytochrome creductase complex chaperone). Association of the above eight SNPs with spine BS were replicated in one Caucasian and one Chinese populations. Meta-analyses (N = 7,416) generated much stronger association signals for these SNPs (e.g., P = 1.86E-07 for SNP rs6060373), supporting association of UQCC with spine BS across ethnicities.
This study identified a novel locus, i.e., the UQCC gene, for spine BS variation in humans. Future functional studies will contribute to elucidating the mechanisms by which UQCC regulates bone growth and development.
doi:10.1016/j.bone.2012.11.028
PMCID: PMC3682469  PMID: 23207799
Spine bone size; GWAS; UQCC
13.  The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells 
In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.
doi:10.1038/cmi.2013.40
PMCID: PMC4002151  PMID: 24037184
DHX33; helicase; innate immunity; IPS-1; myeloid dendritic cell; viral nucleic acid
14.  Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells 
Cancer research  2010;70(23):9581-9590.
CD4+CD25+Foxp3+ T-regulatory cells (Tregs) accumulate in tumors, however little is known about how the tumor environment influences this process. Here we show that human melanomas express ICOS-ligand (ICOS-L/B7H) that can provide costimulation through ICOS for the expansion of activated Tregs maintaining high Foxp3 and CD25 expression as well as suppressive function. Thus, ICOS-L expression by melanoma tumor cells may directly drive Treg activation and expansion in the tumor microenvironment as another mechanism of immune evasion.
doi:10.1158/0008-5472.CAN-10-1379
PMCID: PMC3058814  PMID: 21098714
Melanoma; ICOSL; Treg; Foxp3; ICOS
15.  The Fat Mass and Obesity Associated Gene, FTO, Is Also Associated with Osteoporosis Phenotypes 
PLoS ONE  2011;6(11):e27312.
Obesity and osteoporosis are closely correlated genetically. FTO gene has been consistently identified to be associated with obesity phenotypes. A recent study reported that the mice lacking Fto could result in lower bone mineral density (BMD). Thus, we hypothesize that the FTO gene might be also important for osteoporosis phenotypes. To test for such a hypothesis, we performed an association analyses to investigate the relationship between SNPs in FTO and BMD at both hip and spine. A total of 141 SNPs were tested in two independent Chinese populations (818 and 809 unrelated Han subjects, respectively) and a Caucasian population (2,286 unrelated subjects). Combining the two Chinese samples, we identified 6 SNPs in FTO to be significantly associated with hip BMD after multiple testing adjustments, with the combined P values ranged from 4.99×10−4–1.47×10−4. These 6 SNPs are all located at the intron 8 of FTO and in high linkage disequilibrium. Each copy of the minor allele of each SNP was associated with increased hip BMD values with the effect size (beta) of ∼0.025 and ∼0.015 in the Chinese sample 1 and 2, respectively. However, none of these 6 SNPs showed significant association signal in the Caucasian sample, by presenting some extent of ethnic difference. Our findings, together with the prior biological evidence, suggest that the FTO gene might be a new candidate for BMD variation and osteoporosis in Chinese populations.
doi:10.1371/journal.pone.0027312
PMCID: PMC3220685  PMID: 22125610
16.  Genetic Association Study of Common Mitochondrial Variants on Body Fat Mass 
PLoS ONE  2011;6(6):e21595.
Mitochondria play a central role in ATP production and energy metabolism. Previous studies suggest that common variants in mtDNA are associated with several common complex diseases, including obesity. To test the hypothesis that common mtDNA variants influence obesity-related phenotypes, including BMI and body fat mass, we genotyped a total of 445 mtSNPs across the whole mitochondrial genome in a large sample of 2,286 unrelated Caucasian subjects. 72 of these 445 mtSNPs passed quality control criteria, and were used for subsequent analyses. We also classified all subjects into nine common European haplogroups. Association analyses were conducted for both BMI and body fat mass with single mtSNPs and mtDNA haplogroups. Two mtSNPs, mt4823 and mt8873 were detected to be significantly associated with body fat mass, with adjusted P values of 4.94×10-3 and 4.58×10-2, respectively. The minor alleles mt4823 C and mt8873 A were associated with reduced fat mass values and the effect size (β) was estimated to be 3.52 and 3.18, respectively. These two mtSNPs also achieved nominally significant levels for association with BMI. For haplogroup analyses, we found that haplogroup X was strongly associated with both BMI (adjusted P = 8.31×10-3) and body fat mass (adjusted P = 5.67×10-4) Subjects classified as haplogroup X had lower BMI and fat mass values, with the β estimated to be 2.86 and 6.03, respectively. Our findings suggest that common variants in mitochondria might play a role in variations of body fat mass. Further molecular and functional studies will be needed to clarify the potential mechanism.
doi:10.1371/journal.pone.0021595
PMCID: PMC3126834  PMID: 21747914
17.  A novel subset of CD4+ TH2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma 
The Journal of Experimental Medicine  2010;207(11):2479-2491.
Memory CD4+ T cells that produce both Th2 and Th17 cytokines are increased in the blood of patients with atopic asthma and in the lungs of asthmatic mice, where they contribute to inflammation.
The inflammatory cytokine interleukin (IL)-17 is involved in the pathogenesis of allergic diseases. However, the identity and functions of IL-17–producing T cells during the pathogenesis of allergic diseases remain unclear. Here, we report a novel subset of TH2 memory/effector cells that coexpress the transcription factors GATA3 and RORγt and coproduce TH17 and TH2 cytokines. Classical TH2 memory/effector cells had the potential to produce IL-17 after stimulation with proinflammatory cytokines IL-1β, IL-6, and IL-21. The number of IL-17-TH2 cells was significantly increased in blood of patients with atopic asthma. In a mouse model of allergic lung diseases, IL-17–producing CD4+ TH2 cells were induced in the inflamed lung and persisted as the dominant IL-17–producing T cell population during the chronic stage of asthma. Treating cultured bronchial epithelial cells with IL-17 plus TH2 cytokines induced strong up-regulation of chemokine eotaxin-3, Il8, Mip1b, and Groa gene expression. Compared with classical TH17 and TH2 cells, antigen-specific IL-17–producing TH2 cells induced a profound influx of heterogeneous inflammatory leukocytes and exacerbated asthma. Our findings highlight the plasticity of TH2 memory cells and suggest that IL-17–producing TH2 cells may represent the key pathogenic TH2 cells promoting the exacerbation of allergic asthma.
doi:10.1084/jem.20101376
PMCID: PMC2964570  PMID: 20921287
18.  Genome-wide Association Study of Exercise Behavior in Dutch and American Adults 
Introduction
The objective of this study was to identify genetic variants that are associated with adult leisure-time exercise behavior using genome-wide association in two independent samples.
Methods
Exercise behavior was measured in 1,772 unrelated Dutch and 978 unrelated American adults with detailed questions about type, frequency and duration of exercise. Individuals were classified into regular exercisers or non-exercisers using a threshold of 4 METhours (metabolic equivalents*hours per week). Regular exercisers were further divided into 5 categories of METhours, ranging from moderate (>=4 METhours) to highly vigorous (>=40 METhours) exercisers. Genome-wide association analyses with a total of 470,719 SNPs were conducted in both samples independently using regression-based techniques in SNPtest, including sex, age and BMI as covariates.
Results
SNPs located in SGIP1, DNASE2B, PRSS16, ERCC2 and PAPSS2 were associated with exercise participation (combined p-value between 0.0004 and 4.5*10-6 with the same direction of allelic effects in both samples). Associations of candidate genes based on existing literature were replicated for the LEPR gene in the American sample (rs12405556, p=0.0005) and for the CYP19A1 gene in the Dutch sample (rs2470158, 0.0098).
Conclusion
Two genes (SGIP1 and LEPR) are expressed in the hypothalamus and involved in the regulation of energy homeostasis. Their effects were independent of BMI, suggesting a direct role of hypothalamic factors in the drive to exercise.
doi:10.1249/MSS.0b013e3181a2f646
PMCID: PMC2895958  PMID: 19727025
Physical activity; sports participation; genetics; genotype imputation; energy homeostasis
19.  A NOVEL STRATEGY FOR RAPID AND EFFICIENT ISOLATION OF HUMAN TUMOR-SPECIFIC CD4+ AND CD8+ T-CELL CLONES 
Journal of immunological methods  2007;331(1-2):13-26.
Adoptive therapy with antigen-specific T cells is a promising approach for the treatment of infectious diseases and cancer. However, cloning of antigen-specific T cells by the traditional approach of limiting dilution is a time-consuming, laborious, and inefficient process. Here, we describe a novel flow cytometric strategy for rapid isolation of human tumor antigen-specific T-cell clones by using T-cell receptor (TCR) Vβ antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. The CFSE dilution following antigen stimulation identified proliferating antigen-specific T cells, and the TCRVβ antibodies allowed distinguishing T cells at the clonal level from a heterogeneous T-cell population. This method of TCR Vβ/CFSE dilution was used for the isolation of four different human lymphoma and melanoma-specific CD4+ and CD8+ T-cell clones reactive against defined and undefined tumor antigens. Isolated tumor-specific T-cell clones could be expanded to large numbers ex vivo while maintaining phenotype, function, and tumor antigen specificity. The method was simple, efficient, and reproducible, and may have potential application for the development of adoptive immunotherapeutic strategies.
doi:10.1016/j.jim.2007.09.006
PMCID: PMC2265521  PMID: 17959194
T cells; cloning; TCR; Vbeta; tumor; human; cancer; melanoma; lymphoma
20.  Is Replication the Gold Standard for Validating Genome-Wide Association Findings? 
PLoS ONE  2008;3(12):e4037.
With the advent of genome-wide association (GWA) studies, researchers are hoping that reliable genetic association of common human complex diseases/traits can be detected. Currently, there is an increasing enthusiasm about GWA and a number of GWA studies have been published. In the field a common practice is that replication should be used as the gold standard to validate an association finding. In this article, based on empirical and theoretical data, we emphasize that replication of GWA findings can be quite difficult, and should not always be expected, even when true variants are identified. The probability of replication becomes smaller with the increasing number of independent GWA studies if the power of individual replication studies is less than 100% (which is usually the case), and even a finding that is replicated may not necessarily be true. We argue that the field may have unreasonably high expectations on success of replication. We also wish to raise the question whether it is sufficient or necessary to treat replication as the ultimate and gold standard for defining true variants. We finally discuss the usefulness of integrating evidence from multiple levels/sources such as genetic epidemiological studies (at the DNA level), gene expression studies (at the RNA level), proteomics (at the protein level), and follow-up molecular and cellular studies for eventual validation and illumination of the functional relevance of the genes uncovered.
doi:10.1371/journal.pone.0004037
PMCID: PMC2605260  PMID: 19112512
21.  Relationship of obesity with osteoporosis 
Context
The relationship between obesity and osteoporosis has been widely studied, and epidemiological evidence shows that obesity is correlated with increased bone mass. Previous analyses, however, did not control for the mechanical loading effects of total body weight on bone mass and may have generated a confounded or even biased relationship between obesity and osteoporosis.
Objective
To re-evaluate the relationship between obesity and osteoporosis by accounting for the mechanical loading effects of total body weight on bone mass.
Methods
We measured whole body fat mass, lean mass, percentage fat mass (PFM), body mass index (BMI), and bone mass in two large samples of different ethnicity: 1,988 unrelated Chinese subjects and 4,489 Caucasian subjects from 512 pedigrees. We first evaluated the Pearson correlations among different phenotypes. We then dissected the phenotypic correlations into genetic and environmental components, with bone mass unadjusted, or adjusted, for body weight. This allowed us to compare the results with and without controlling for mechanical loading effects of body weight on bone mass.
Results
In both Chinese and Caucasians, when the mechanical loading effect of body weight on bone mass was adjusted for, the phenotypic correlation (including its genetic and environmental components) between fat mass (or PFM) and bone mass was negative. Further multivariate analyses in subjects stratified by body weight confirmed the inverse relationship between bone mass and fat mass, after mechanical loading effects due to total body weight was controlled.
Conclusions
Increasing fat mass may not have a beneficial effect on bone mass.
doi:10.1210/jc.2006-0572
PMCID: PMC1868430  PMID: 17299077
22.  TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells 
Nature medicine  2005;11(6):653-660.
Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, since in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+CD16+ macrophages and CD1b+DC-SIGN− dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of IL-15/IL-15R. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of GM-CSF/GM-CSFR, promoted T cell activation and secreted proinflammatory cytokines. While DC-SIGN+ macrophages were detected in lesions of all leprosy patients, CD1b+ dendritic cells were not detected in patients with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by Th1 responses. In T-lep lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells appears critically to influence effective host defenses in human infectious disease.
doi:10.1038/nm1246
PMCID: PMC1409736  PMID: 15880118
23.  Patterns of linkage disequilibrium and haplotype distribution in disease candidate genes 
BMC Genetics  2004;5:11.
Background
The adequacy of association studies for complex diseases depends critically on the existence of linkage disequilibrium (LD) between functional alleles and surrounding SNP markers.
Results
We examined the patterns of LD and haplotype distribution in eight candidate genes for osteoporosis and/or obesity using 31 SNPs in 1,873 subjects. These eight genes are apolipoprotein E (APOE), type I collagen α1 (COL1A1), estrogen receptor-α (ER-α), leptin receptor (LEPR), parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1), transforming growth factor-β1 (TGF-β1), uncoupling protein 3 (UCP3), and vitamin D (1,25-dihydroxyvitamin D3) receptor (VDR). Yin yang haplotypes, two high-frequency haplotypes composed of completely mismatching SNP alleles, were examined. To quantify LD patterns, two common measures of LD, D' and r2, were calculated for the SNPs within the genes. The haplotype distribution varied in the different genes. Yin yang haplotypes were observed only in PTHR1 and UCP3. D' ranged from 0.020 to 1.000 with the average of 0.475, whereas the average r2 was 0.158 (ranging from 0.000 to 0.883). A decay of LD was observed as the intermarker distance increased, however, there was a great difference in LD characteristics of different genes or even in different regions within gene.
Conclusion
The differences in haplotype distributions and LD patterns among the genes underscore the importance of characterizing genomic regions of interest prior to association studies.
doi:10.1186/1471-2156-5-11
PMCID: PMC421754  PMID: 15157284
linkage disequilibrium (LD); haplotype; single nucleotide polymorphism (SNP)
24.  Current limitations of SNP data from the public domain for studies of complex disorders: a test for ten candidate genes for obesity and osteoporosis 
BMC Genetics  2004;5:4.
Background
Public SNP databases are frequently used to choose SNPs for candidate genes in the association and linkage studies of complex disorders. However, their utility for such studies of diseases with ethnic-dependent background has never been evaluated.
Results
To estimate the accuracy and completeness of SNP public databases, we analyzed the allele frequencies of 41 SNPs in 10 candidate genes for obesity and/or osteoporosis in a large American-Caucasian sample (1,873 individuals from 405 nuclear families) by PCR-invader assay. We compared our results with those from the databases and other published studies. Of the 41 SNPs, 8 were monomorphic in our sample. Twelve were reported for the first time for Caucasians and the other 29 SNPs in our sample essentially confirmed the respective allele frequencies for Caucasians in the databases and previous studies. The comparison of our data with other ethnic groups showed significant differentiation between the three major world ethnic groups at some SNPs (Caucasians and Africans differed at 3 of the 18 shared SNPs, and Caucasians and Asians differed at 13 of the 22 shared SNPs). This genetic differentiation may have an important implication for studying the well-known ethnic differences in the prevalence of obesity and osteoporosis, and complex disorders in general.
Conclusion
A comparative analysis of the SNP data of the candidate genes obtained in the present study, as well as those retrieved from the public domain, suggests that the databases may currently have serious limitations for studying complex disorders with an ethnic-dependent background due to the incomplete and uneven representation of the candidate SNPs in the databases for the major ethnic groups. This conclusion attests to the imperative necessity of large-scale and accurate characterization of these SNPs in different ethnic groups.
doi:10.1186/1471-2156-5-4
PMCID: PMC395827  PMID: 15113403
SNP Databases; Polymorphism; Obesity; Osteoporosis; Complex diseases; Ethnicity
25.  Targeting of Toll-like receptors inhibits CD4+ regulatory T cell function and activates lymphocytes in human PBMCs 
Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs) thus preventing the development of durable anti-tumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that Toll-like receptor ligands (TLRL) could modulate the function of these T-cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we employed a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4+, CD8+, Tregs), B cells and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8+ T cells, B cells and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS and TLR8/7L:CL075 also blocked Treg suppression of CD4+ or CD8+ T cell proliferation but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075 and TLR9L:CpG-(A-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4+ T cells proliferation; 2) inhibit the expansion of IL-10+ nTregs and induction of IL-10+ CD4+ Tregs (Tr1); and 3) block nTreg suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.
doi:10.4049/jimmunol.1203334
PMCID: PMC4347808  PMID: 24928999

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